Modulation of the ligand-field anisotropy in a series of ferric low-spin cytochrome c mutants derived from Pseudomonas aeruginosa cytochrome c-551 and Nitrosomonas europaea cytochrome c-552: a nuclear magnetic resonance and electron paramagnetic resonance study.
Cytochromes of the c type with histidine-methionine (His-Met) heme axial ligation play important roles in electron-transfer reactions and in enzymes. In this work, two series of cytochrome c mutants derived from Pseudomonas aeruginosa (Pa c-551) and from the ammonia-oxidizing bacterium Nitrosomonas europaea (Ne c-552) were engineered and overexpressed. In these proteins, point mutations were induced in a key residue (Asn64) near the Met axial ligand; these mutations have a considerable impact both on heme ligand-field strength and on the Met orientation and dynamics (fluxionality), as judged by low-temperature electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectra. Ne c-552 has a ferric low-spin (S = 1/2) EPR signal characterized by large g anisotropy with g(max) resonance at 3.34; a similar large g(max) value EPR signal is found in the mitochondrial complex III cytochrome c1. In Ne c-552, deletion of Asn64 (NeN64Delta) changes the heme ligand field from more axial to rhombic (small g anisotropy and g(max) at 3.13) and furthermore hinders the Met fluxionality present in the wild-type protein. In Pa c-551 (g(max) at 3.20), replacement of Asn64 with valine (PaN64V) induces a decrease in the axial strain (g(max) at 3.05) and changes the Met configuration. Another set of mutants prepared by insertion (ins) and/or deletion (Delta) of a valine residue adjacent to Asn64, resulting in modifications in the length of the axial Met-donating loop (NeV65Delta, NeG50N/V65Delta, PaN50G/V65ins), did not result in appreciable alterations of the originally weak (Ne c-552) or very weak (Pa c-551) axial field but had an impact on Met orientation, fluxionality, and relaxation dynamics. Comparison of the electronic fingerprints in the overexpressed proteins and their mutants reveals a linear relationship between axial strain and average paramagnetic heme methyl shifts, irrespective of Met orientation or dynamics. Thus, for these His-Met axially coordinated Fe(III), the large g(max) value EPR signal does not represent a special case as is observed for bis-His axially coordinated Fe(III) with the two His planes perpendicular to each other.
Zoppellaro
Giorgio
G
Department of Molecular Biosciences, University of Oslo, Post Office Box 1041 Blindern, Oslo NO-0316, Norway.
Harbitz
Espen
E
Kaur
Ravinder
R
Ensign
Amy A
AA
Bren
Kara L
KL
Andersson
K Kristoffer
KK
D000595:Amino Acid Sequence; D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008745:Methylation; D008872:Microwaves; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D043370:Nitrosomonas europaea / Q000201:enzymology* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D008024:Ligands; D006418:Heme; C017167:cytochrome C(551); C022302:cytochrome C-552